BME100 f2013:W900 Group13 L4

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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Contents

OUR TEAM

Name: Allison MarleyRole(s): Research And Development
Name: Allison Marley
Role(s): Research And Development
Name: Tyler AngellRole(s): Initial Machine Testing
Name: Tyler Angell
Role(s): Initial Machine Testing
Name: Cory RieckenRole(s): Initial Machine Testing
Name: Cory Riecken
Role(s): Initial Machine Testing
Name: Reem GeraisRole(s):  Protocol Planning Specialist
Name: Reem Gerais
Role(s): Protocol Planning Specialist
Name: Andrew LucRole(s): Protocol Planning Specialist
Name: Andrew Luc
Role(s): Protocol Planning Specialist

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
Image:allisonopenpcr.jpgImage:openPCR.jpg
The OpenPCR machine runs PCR reactions and can be monitored by a computer to which the machine can be connected with a USB 2.0 cable. The machine has a heated lid which prevents condensation on the caps of the PCR tubes. If this part did not work, water would condensate on the cap and cause the salt concentration to be too high for the polymerase to function properly. The heated lid is situated over the 16 tube PCR block, which holds and heats the samples in the PCR tubes. If the block did not work, the samples would not be heated to the necessary temperatures and there would not be a PCR reaction. Information about the reaction is displayed on the screen located on top of the box. Inside the box there are the heating components for the PCR block. These heating components go to a heat sink which leads into an air filter which a fan moves air through so as to keep the temperature within the box from getting too hot and the unit from overheating. The wiring for the PCR block, the cooling system, and the display are all hooked to a motherboard situated at the bottom of the box. The motherboard controls the temperature through a connection to the PCR block and runs the OpenPCR machine.
Images from:
http://openwetware.org/wiki/BME103:T130_Group_10
http://www.forbes.com/sites/tedgreenwald/2011/12/31/dna-sequencing-for-fun-and-profit-a-low-cost-platform-for-garage-biotech/

Experimenting With the Connections

When we unplugged (part 3) from either end, the machine's screen turned off. When we plugged the cord back in, the screen turned back on. We believe this part supplies power to the screen.

When we unplugged the white wire that connects (part 6) to (part 2), the machine showed no noticeable change. However, we believe that the temperature controls for the thermocycler will not work when the white wire is disconnected.

Test Run Test Date: 10/23/13 Experience: The Open PCR's time functioned properly, but the component that keeps track of the cycles did not. The unit indicated that it was on the first cycle of thirty-five throughout the duration of the PCR reaction. (Write the date you first tested Open PCR and your experience(s) with the machine)



Protocols

Thermal Cycler Program
The Thermal Cycler Program:

1. Begins with the initial denaturation step which consists of one cycle at 95C for three minutes

2. Moves next to the denaturation step which consists of 35 cycles at 95C for 30 seconds, the anneal step which includes 57C for 30 seconds, and the extended step including 72C for 30 seconds for each cycle.

3. The Final extension step occurs at 72C for 3 minutes

4. The refrigeration of the DNA occurs during the final hold at 4C


DNA Sample Set-up
Table #1

(C+) Tube

Positive Control: cancer DNA Template

Tube (1A)

Patient 1 ID: 26734

Tube (1B)

Patient 1 ID: 26734

Tube (1C)

Patient 1 ID:26734

(C-) Tube

Negative Control: Non-cancer DNA template

Tube (2A)

Patient 2 ID:22707

Tube (2B)

Patient 2 ID:22707

Tube (2C)

Patient 3 ID:22707


DNA Sample Set-up Procedure

1. Step 1: Label each tube with its respective contents to prevent misdiagnosing patients. (Refer to Table #1 for labelling)
2. Step 2: Use pipettes to dispense 50 μL of the PCR reaction mixes into each of the 8 tubes.
3. Step 3: Using different disposable pipette tips to dispense 50 μL of each DNA sample into their respective tubes
4. Step 4: Be sure to change the pipette tips with each use in order to prevent cross-contamination of the samples
5. Step 5: After all of the tubes contain both the PCR Reaction Mix and the DNA/Primer Mix, place the tubes into the PCR machine and close the lid.
6. Step 6: Make sure the PCR machine is programmed with the correct cycles and temperatures
7. Step 7: Connect the PCR machine to the computer and make sure the power cord is connected to the machine.
8. Step 8: Run the Open PCR software on the computer with the Thermal Cycler program


PCR Reaction Mix
Each of the 8 tubes will contain 50 μL of the reaction mix. The PCR reaction mixes will contain each of the following:

 •Taw DNA Polymerase
 •MgCl2
 •dNTP


DNA/ primer mix
In addition to the PCR reaction mix, each tube will contain 50 μL of the following:

 •A Forward Primer
 •A Reverse Primer

Six of the eight tubes will contain unique DNA samples from two different patients. The remaining two tubes will contain control templates with and without cancerous DNA: the Positive Control Tube will contain the cancerous DNA and the Negative will not.





Research and Development

PCR - The Underlying Technology
Function of Components in PCR reaction
The template DNA is the strand of DNA that is used to create a complementary strand. This DNA strand is used to double to amount of DNA per cycle. After the DNA is placed in the test tube, the primers are added. Since the primers have any sequence of nucleotides desired, they can then attach to sites on the DNA strands that are at either end of the segment you want to copy. The primers are powerful for copying specific DNA sequences since there is no chance they will target the wrong site. After the primers are added, the Taq DNA polymerase is added to the test tube. Taq polymerase functions to copy a cell's DNA before it divides into two seperate strands. When Taq polymerase reaches a primer that's base paired with a longer piece of DNA, it attaches itself and starts adding primers. MgCl2 is required fro Taq polymerase to function effectively. When th enzyme polymerase binds to the DNA strand, it requires Mg+ ions with OH- groups to remove a hydrogen proton from the deoxyribose of th enucleotide in order to add the next nucleotide. The deoxyribonucleotides (dNTP's) are then added. The dNTP's are single units of the bases A,T,G,and C. These bases are then used as genetic building blocks to create billions of DNA copies.Adenine (A) will pair with Thymine (T) ans Guanine (G) will pair with Cytosine (C).
Thermal Cycling
The initial step of PCR occers at 95 degrees celcius for 3 minutes. In this stage the initial denaturation occurs, with an additional 30 seconds the DNA double helix separates, creating two-single stranded DNA molecules. Next the temperature is lowered to 57 degrees celcius for a period of 30 seconds. During the annealing stage, single stranded DNA molecules naturally attempt to pair up but there are much more primer sequences than DNA. The primers crowd their way in and lock onto their target before the strands have a chance to rejoin. Next, at 72 degrees celcius for 30 seconds, the DNA polymerase is activated and locates a primer attached to a single DNA strand. It begins to add complementary nucleotides onto the strand. A final step at 72 degrees celcius for 3 minutes occurs where a final extension period occurs during this time. A final hold then takes place at 4 degrees celcius during this time the cylcle refrigerates the DNA for a final hold. This minimizes any polymerase activity that might occur at higher temperatures.





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